PROJECT SUMMARY/ABSTRACT DNA double-strand breaks (DSBs) are among the most toxic DNA lesions because failure to repair them accurately can lead to large-scale genome rearrangements. Homologous recombination (HR) is a major, conserved pathway by which cells repair DSBs. It functions by locating a homologous sequence and using it as a template for DNA synthesis, yielding a highly accurate repair outcome. The importance of HR is underscored by the fact that mutations in a number of HR genes, including the tumor suppressors BRCA1 and BRCA2, are associated with increased cancer risk. Rare genetic cancer predisposition syndromes are also associated with aberrant or defective HR. Mutations in the BLM helicase, which participates in the initiation of HR as well as resolution of DNA structures formed late in the process, cause Bloom Syndrome. Fanconi Anemia is caused by mutations in a large complex of ?FANC? proteins that detect DNA interstrand crosslinks, lesions that are first excised before being converted into DSBs and routed into the HR pathway. Recent research has uncovered an intriguing link between DSB repair and the RNAi pathway. Specifically, small RNAs have been identified at DSB sites, where they are important for cell cycle checkpoint activation (1) and also HR (2, 3). These RNAs are produced by Dicer and processed by the Argonaute family protein Ago2. Cell-based studies have shown that they are required for the optimal recruitment of RAD51 to DSB sites, but not for upstream steps in HR (2). RAD51 is the recombinase responsible for catalyzing the HR reaction. BRCA2 loads RAD51 onto ssDNA tails formed at the break site, where RAD51 forms a filament that then searches for a homologous DNA template to initiate repair. The mechanism by which small RNAs promote RAD51 loading is unknown. We have developed unique reconstituted systems to examine the steps in HR, including RAD51-ssDNA nucleoprotein filament assembly. In this project, we will apply our expertise to characterize the mechanism by which small RNAs function in HR. In Aim 1, we will purify Ago2, investigate its interaction with RAD51, and generate mutants defective for the interaction. In Aim 2, our unique biochemical systems will be used to pinpoint the role of Ago2 and its associated small RNAs in loading RAD51. The importance of base pairing between the small RNAs and the ssDNA tails onto which RAD51 loads will be assessed, and mutants generated in Aim 1 will be tested. Interplay with DSS1, a BRCA2-interacting protein that assists in removing RPA from the ssDNA to make way for RAD51, will also be investigated. Besides characterizing the novel role of small RNAs in HR, this project will contribute to our mechanistic understanding of a BRCA2-dependent step in HR, and will therefore provide insights into the initial events that lead to tumorigenesis in the breast, ovaries and other organs.